Determination by automated instrumentation of blood, pH, PO2, PCO2, electrolytes, metabolites and CO-Oximetry fractions are routine clinical laboratory analyses. CO-Oximeter instruments typically measure total hemoglobin concentration (tHb), and hemoglobin fractions e.g., oxyhemoglobin (O2Hb), methemoglobin (MetHb), carboxyhemoglobin (COHb), sulfhemoglobin (SHb), and deoxyhemoglobin (HHb). These fractions are referred to as CO-Ox fractions or hemoglobin fractions or hemoglobin derivatives. Currently available instruments such as IL 682 and GEM® Premier™ 4000 (Instrumentation Laboratory Company, Bedford, Mass.) have the ability to measure blood pH, gases, electrolytes, metabolites, total bilirubin, and/or total hemoglobin and hemoglobin fractions in the same instrument.
Blood hemoglobin and hemoglobin fractions absorb visible light within the wavelength range of 450-700 nm. For example, a normal oxygenated blood spectrum has two main peak wavelengths at 542 and 578 nm and absorbance rapidly decreases close to zero at wavelengths greater than 610 nm.
Based on the known hemoglobin fraction wavelength regions and the known relationship between hemoglobin concentration and absorbance spectrum, CO-Oximeters analyze blood samples by comparing the sample absorbance spectrum to an instrument calibration set consisting of hemoglobin spectra of known concentrations (FIG. 1) of each individual fraction. The data are analyzed by multicomponent analysis to calculate the concentrations of total hemoglobin and each hemoglobin fraction present in a patient's blood sample.
Reference materials generally function to validate the performance of a diagnostic instrument, such as a CO-Oximeter. These materials are typically aqueous or blood-based materials. As used herein, blood-based materials are materials derived from human or animal blood that include some component of red blood cells, but not referring to plasma or serum materials without some component of red blood cells. The blood-based materials allow for the direct match of the spectrum of blood. However, there are no blood-based quality control (QC) or reference materials available that include both hemoglobin fractions and bilirubin due to limited useful shelf-life caused by, for example, instability of bilirubin and/or bilirubin. Traditionally, bilirubin is validated using plasma or serum based reference materials that do not include hemoglobin. Therefore, reference materials for bilirubin on whole/lysed blood CO-Oximetry systems are limited in their ability to establish corrections for hemoglobin interference due to the lack of bilirubin reference materials that include hemoglobin and hemoglobin fractions.
Bilirubin, a principal component of bile pigment in a body fluid such as blood, is produced by the decomposition of heme from hemoglobin in red blood cells (RBCs). Two main fractions of bilirubin are present in blood. One is free or unconjugated bilirubin (indirect) and the other is conjugated (direct) bilirubin. Conjugated bilirubin is chemically bound to glucuronic acid to render water solubility of bilirubin for excretion from the body.
Increased serum bilirubin levels, a combination of direct/conjugated and indirect/unconjugated bilirubin, produce the clinical condition known as jaundice. The liver converts bilirubin to conjugated bilirubin so that it is excreted from the body. In obstructive jaundice and in liver disease, excretion or metabolism is impaired and an elevated conjugated bilirubin fraction occurs. This form of jaundice differs from the cause of neonatal jaundice. The liver of newborns is not fully developed at birth, and newborns often lack the enzymes necessary to convert the unconjugated form to the conjugated form of bilirubin for excretion. Increased indirect bilirubin is an important measurement for newborns.
Traditional clinical laboratory measurements to assay total bilirubin in blood use the Jendrassik-Gróf principle, where all bilirubin species react with diazonated sulfanilic acid, in the presence of caffeine-benzoate acting as a promoter, to yield a red azopyrole. On addition of alkaline tartrate to serum or plasma, the color turns blue, which has a maximum absorbance at 598 nm. A disadvantage of this prior art method is that it cannot be completed using whole blood. Serum or plasma must be used, which requires separation from cellular blood components, typically by centrifugation, before measurement. More recently, methods have been developed whereby bilirubin is measured via direct spectrophotometry on whole blood using analytical instruments, for example, the GEM Premier 4000 cartridge-based system (Instrumentation Laboratory Company, Bedford, Mass.) or the IL Synthesis (Instrumentation Laboratory Company, Bedford, Mass.).
The increasing popularity of the use of direct spectrophotometry in clinical laboratories can be attributed to ease of use, speed of results, and convenience. These systems measure whole blood, thereby eliminating the time required to separate blood formed elements from plasma or serum, using spectrometric measurements to quantify bilirubin, total hemoglobin concentration (tHb), and the hemoglobin fractions O2Hb, HHb, COHb, SHb, and MetHb, i.e., the CO-Ox fractions, and are often coupled to electrochemical sensors to quantify other analytes of interest. Some clinical analyzers only report on a portion of these analytes. CO-Oximeters often lyse RBCs by chemical means, mechanical means, or both, to reduce scattering effects of cell membranes and to improve overall accuracy of the measuring system by reducing the background noise.
Bilirubin, unlike many other blood constituents, is highly unstable in many reference materials because it is sensitive to light, oxygen, and ambient temperatures. For this reason bilirubin based reference materials typically require refrigerated or frozen storage conditions. In the presence of either light, elevated temperature, or oxygen, the conversion of bilirubin to biliverdin, an analyte which has a very different spectral absorbance than bilirubin, is accelerated. For example, in current blood-based reference materials, such as those disclosed in U.S. Pat. No. 4,485,174 i.e., blood-based materials that contain hemoglobin, oxygen is required in order to provide stable, clinically meaningful levels of oxyhemoglobin, the primary fraction of total hemoglobin. The presence of oxygen accelerates the conversion of bilirubin to biliverdin. To minimize the effect of bilirubin's instability, i.e., the conversion of bilirubin to a compound, such as biliverdin, having a different absorbance, prior art reference materials for bilirubin, e.g., those used for CAP NB-surveys, are typically dispensed in vials and stored frozen. This storage method is unsatisfactory because it increases measurement variability due to rigorous handling procedures and provides an opportunity for pre-analytical error. Furthermore, these materials do not provide for measurement of total hemoglobin and/or hemoglobin fractions together with bilirubin in the same reference material in which the quantities of these analytes relate to meaningful clinical levels. Freezing and thawing, as required for many bilirubin reference materials, is unsatisfactory for hemoglobin based reference materials. The risk of an inaccurate measurement is elevated when hemoglobin is measured after freezing because the temperature change may induce conversion of oxyhemoglobin to methemoglobin.
At present, because of the instability of solutions containing bilirubin stored at refrigerated temperatures and the instability of hemoglobin materials upon freezing and thawing, a single blood based quality control or reference material for clinically meaningful concentrations of bilirubin, total hemoglobin, and its fractions, has not been developed. Organic dye based colored materials are in use by some manufacturers to simulate the spectral properties of hemoglobin and bilirubin assays. Such dye based products are useful for general quality check of the optical systems but are not very specific in qualifying or identifying performance quality issues. In addition, dye based quality materials require manufacturer-specific secondary correction factors or analytical algorithms separate from those used to analyze clinical samples to report clinically meaningful hemoglobin or bilirubin results. For this reason, a single blood based reference material, i.e. a blood-based material other than plasma or serum, that provides clinically relevant measurements of both hemoglobin and bilirubin independent of manufacturer or analytical system has a potential for clinical and commercial interest.
In other words, in spite of the efficiency in qualifying and calibrating instrument systems that would be provided by such a single reference material and the long felt commercial need for such a product, a single blood based reference material providing clinically relevant concentrations of bilirubin, hemoglobin and its fractions, without requiring instrument platform-specific secondary correction factors or analytical algorithms, is not available.
Currently, two separate materials are required for calibrating or running quality control on bilirubin, total hemoglobin, and hemoglobin fractions; one for bilirubin and one for total hemoglobin and hemoglobin fractions. Accordingly, the laboratory time required for conducting quality control on bilirubin and hemoglobin is longer than would be required with a single reference material that includes bilirubin, total hemoglobin, and hemoglobin fractions. In addition, evaluation of bilirubin in the presence of clinically relevant concentrations of hemoglobin provides more accurate corrections for hemoglobin interference on bilirubin, and a more accurate evaluation of an analyzer's ability to accurately measure these analytes in patient samples.